Method for the determination of α-ketoisocaproate in biological samples using D-α-hydroxyisocaproic dehydrogenase from lactobacillus casei

Abstract

The selective determination of alpha -ketoisocaproate in samples of body fluids, especially in serum, is achieved, without previous separation, directly in the fluid and in the presence of NADH, by means of a KIC-specific D- alpha -hydroxy-isocaproic dehydrogenase that originates specifically from strains of Lactobacillus casei producing this enzyme, in particular strains of Lactobacillus casei subspecies pseudoplantarum or casei. The KIC determination is used, in particular, for diagnosing maple syrup urine disease. Expedient for this purpose are analytical kits which have a divisible stock of D- alpha -hydroxyisocaproic dehydrogenase from Lactobacillus casei, in particular from the Lactobacillus casei (DSM 20008).

What is claimed is: 
     
       1. A method for the selective determination of α-ketoisocaproate content in a sample, comprising the steps of: (A) providing a sample of deproteinized serum or urine from a subject;   (B) bringing said sample, in the presence of NADH, into contact with a D-α-hydroxyisocaproic dehydrogenase enzyme that selectively catalyzes conversion of α-ketoisocaproate to α-hydroxyisocaproate, said enzyme being obtained from Lactobacillus casein subspecies pseudoplantarum or casei to produce a sample mixture; and then   (C) measuring said sample for occurrence of said conversion by measuring a decrease in NADH.   
     
     
       2. A method as claimed in claim 1, wherein said sample is deproteinized serum. 
     
     
       3. A method as claimed in claim 4, wherein said enzyme is isolated from a strain of Lactobacillus casei subspecies pseudoplantarum deposited under accession number DSM 20008. 
     
     
       4. A method as claimed in claim 2, wherein the decrease in NADH is measured spectrometrically. 
     
     
       5. A method as claimed in claim 2, wherein the sample mixture contains a substrate that consumes NAD +  produced when the α-ketoisocaproate is converted to α-hydroxyisocaproate, said substrate generating a detectable species as it consumes NAD + , the decrease in NADH being measured by detecting said species. 
     
     
       6. A method as claimed in claim 5, wherein the decrease in NADH generates a detectable species which is measured colorimetrically.